The TRAP/Mediator complex contains at least 18 different subunits and plays an essential role in transcriptional activation by nuclear hormone receptors (NRs) as well as a broad range of other activators. A single subunit, TRAP220, acts as a pivotal signaling mediator of cellular growth and development in that it targets TRAP/Mediator to NRs and to the GATA family of transcription factors. TRAP220 is also thought to play an important structural role in terms of holocomplex assembly and stability, and may further act as a binding surface for other essential coregulatory factors. Accordingly, TRAP220 ablation in mice is embryonic lethal. We recently found that TRAP220 is inducibly phosphorylated in vivo and suspect that its functional activity is regulated by such events. We further hypothesize that TRAP220 contains as of yet uncharacterized regulatory domains critical for its coactivator function. The long-term objectives of this proposal are to clearly understand how TRAP220 becomes phosphorylated and how phosphorylation regulates TRAP220 activity in vivo. Furthermore, we seek to more precisely delineate TRAP220's intrinsic functional domains in terms of holocomplex assembly and transcriptional activation. Our specific goals are: 1) To identify the mechanism(s) of TRAP220 phosphorylation in vivo and to determine the precise location of TRAP220 phosphorylation sites. Specific TRAP220 PO4-sites and relevant kinases will be determined using electromobility shift assays, 32-po4-incorporation studies, and mass spectrometry. The specific PO4-sites and relevant kinases will be confirmed via mutagenesis and in vitro kinase reactions. 2) To investigate the functional consequences of TRAP220 phosphorylation. TRAP220 PO4-site mutants (per aim 1) will be tested for defective NR-binding, transcriptional coactivation, subcellular localization and colocalization with other cofactors. We will also carry out cell synchronization studies to examine whether TRAP220 displays cell cycle-dependent phosphorylation. 3) To define the structural elements of TRAP220 necessary for holocomplex assembly and coactivator function. TRAP220 deletion and site-directed mutants will be tested for loss of coactivation by transient transfection in cultured cells and by in vitro transcription assays with chromatin templates. Stably expressed TRAP220 deletion mutants will also be used to biochemically identify specific TRAP220-associated TRAP/Mediator subunits. Given the global importance of TRAP/Mediator, not only for NR signaling but also for transactivation by other regulatory factors, the studies here should have important implications for the transcription field in general. Significantly, these studies may also help to identify and define new targets for therapeutic agents in the treatment of hormone-dependent cancers.